This invention relates to novel nucleoside or nucleotide analogs, and processes for their synthesis and incorporation into polynucleotides.
The following is a brief description of nucleoside analogs. This summary is not meant to be complete but is provided only for an understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Nucleoside modifications of bases and sugars, have been discovered in a variety of naturally occurring RNA (e.g., tRNA, mRNA, rRNA; reviewed by Hall, 1971 The Modified Nucleosides in Nucleic Acids, Columbia University Press, New York; Limbach et al., 1994 Nucleic Acids Res. 22, 2183). In an attempt to understand the biological significance, structural and thermodynamic properties, and nuclease resistance of these nucleoside modifications in nucleic acids, several investigators have chemically synthesized nucleosides, nucleotides and phosphoramidites with various base and sugar modifications and incorporated them into oligonucleotides.
Uhlmann and Peyman, 1990, Chem. Reviews 90, 543, review the use of certain nucleoside modifications to stabilize antisense oligonucleotides.
Usman et al., International PCT Publication Nos. WO/93/15187; and WO 95/13378; describe the use of sugar, base and backbone modifications to enhance the nuclease stability of enzymatic nucleic acid molecules.
Eckstein et al., International PCT Publication No. WO 92/07065 describe the use of sugar, base and backbone modifications to enhance the nuclease stability of enzymatic nucleic acid molecules.
Grasby et al., 1994, Proc. Indian Acad. Sci., 106, 1003, review the xe2x80x9capplications of synthetic oligoribonucleotide analogues in studies of RNA structure and functionxe2x80x9d.
Eaton and Pieken, 1995, Annu. Rev. Biochem., 64, 837, review sugar, base and backbone modifications that enhance the nuclease stability of RNA molecules.
Rosemeyer et al., 1991, Helvetica Chem. Acta, 74, 748, describe the synthesis of 1-(2xe2x80x2-deoxy-xcex2-D-xylofuranosyl) thymine-containing oligodeoxynucleotides.
Seela et al., 1994, Helvetica Chem. Acta, 77, 883, describe the synthesis of 1-(2xe2x80x2-deoxy-xcex2-D-xylofuranosyl) cytosine-containing oligodeoxynucleotides.
Seela et al, 1996, Helvetica Chem. Acta, 79, 1451, describe the synthesis xylose-DNA containing the four natural bases.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the synthesis of xylofuranosyl nucleoside phosphoamidites and polynucleotides comprising such nucleotide modifications of the instant invention.
This invention relates to a compound having the Formula I: 
wherein, R1 is OH, Oxe2x80x94R3, where R3 is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester; Cxe2x80x94R3, where R3 is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester; halo, NHR4 (R4=alkyl (C1-22), acyl (C1-22), substituted or unsubstituted aryl), or OCH2SCH3 (methylthiomethyl), ONHR5 where R5 is independently H, aminoacyl group, peptidyl group, biotinyl group, cholesteryl group, lipoic acid residue, retinoic acid residue, folic acid residue, ascorbic acid residue, nicotinic acid residue, 6-aminopenicillanic acid residue, 7-aminocephalosporanic acid residue, alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide or ester, ON=R6, where R6 is independently pyridoxal residue, pyridoxal-5-phosphate residue, 13-cis-retinal residue, 9-cis-retinal residue, alkyl, alkenyl, alkynyl, alkylaryl, carbocyclic alkylaryl, or heterocyclic alkylaryl; B is independently a nucleotide base or its analog or hydrogen; X is independently a phosphorus-containing group; and R2 is independently blocking group or a phosphorus-containing group.
Specifically, an xe2x80x9calkylxe2x80x9d group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxy, cyano, alkoxy, NO2 or N(CH3)2, amino, or SH.
The term xe2x80x9calkenylxe2x80x9d group refers to unsaturated hydrocarbon groups containing at least one carbonxe2x80x94carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO2, halogen, N(CH3)2, amino, or SH.
The term xe2x80x9calkynylxe2x80x9d refers to an unsaturated hydrocarbon group containing at least one carbonxe2x80x94carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2 or N(CH3)2, amino or SH.
An xe2x80x9carylxe2x80x9d group refers to an aromatic group which has at least one ring having a conjugated xcfx80 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) on aryl groups are halogen, trihalomethyl, hydroxyl, SH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
An xe2x80x9calkylarylxe2x80x9d group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
xe2x80x9cCarbocyclic arylxe2x80x9d groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
xe2x80x9cHeterocyclic arylxe2x80x9d groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
An xe2x80x9camidexe2x80x9d refers to an xe2x80x94C(O)xe2x80x94NHxe2x80x94R, where R is either alkyl, aryl, alkylaryl or hydrogen.
An xe2x80x9cesterxe2x80x9d refers to an xe2x80x94C(O)xe2x80x94ORxe2x80x2, where R is either alkyl, aryl, or alkylaryl.
A xe2x80x9cblocking groupxe2x80x9d is a group which is able to be removed after polynucleotide synthesis and/or which is compatible with solid phase polynucleotide synthesis.
A xe2x80x9cphosphorus containing groupxe2x80x9d can include phosphorus in forms such as dithioates, phosphoramidites and/or as part of an oligonucleotide.
In a preferred embodiment, the invention features a process for synthesis of the compounds of formula I.
In a preferred embodiment the invention features a process for the synthesis of a xylofuranosyl nucleoside phosphoramidite comprising the steps of: a) oxidation of a 2xe2x80x2 and 5xe2x80x2-protected ribonucleoside with a an oxidant such as chromium oxide/pyridine/aceticanhydride, dimethylsulfoxide/aceticanhydride, or Dess-Martin reagent (periodinane) followed by reduction with a reducing agent such as, triacetoxy sodium borohydride, sodium borohydride, or lithium borohydride, under conditions suitable for the formation of 2xe2x80x2 and 5xe2x80x2-protected xylofuranosyl nucleoside; b) phosphitylation under conditions suitable for the formation of xylofuranosyl nucleoside phosphoramidite.
In yet another preferred embodiment, the invention features the incorporation of the compounds of Formula I into polynucleotides. These compounds can be incorporated into polynucleotides enzymatically. For example by using bacteriophage T7 RNA polymerase, these novel nucleotide analogs can be incorporated into RNA at one or more positions (Milligan et al., 1989, Methods Enzymol., 180, 51). Alternatively, novel nucleoside analogs can be incorporated into polynucleotides using solid phase synthesis (Brown and Brown, 1991, in Oligonucleotides and Analogues: A Practical Approach, p. 1, ed. F. Eckstein, Oxford University Press, New York; Wincott et al., 1995, Nucleic Acids Res., 23, 2677; Beaucage and Caruthers, 1996, in Bioorganic Chemistry: Nucleic Acids, p 36, ed. S. M. Hecht, Oxford University Press, New York).
The compounds of Formula I can be used for chemical synthesis of nucleotide-tri-phosphates and/or phosphoramidites as building blocks for selective incorporation into oligonucleotides. These oligonucleotides can be used as an antisense molecule, 2-5A antisense chimera, triplex forming oligonucleotides (TFO) or as an enzymatic nucleic acid molecule. The oligonucleotides can also be used as probes or primers for synthesis and/or sequencing of RNA or DNA.
The compounds of the instant invention can be readily converted into nucleotide diphosphate and nucleotide triphosphates using standard protocols (for a review see Hutchinson, 1991, in Chemistty of Nucleosides and Nucleotides, v.2, pp 81-160, Ed. L. B. Townsend, Plenum Press, New York, USA; incorporated by reference herein).
The compounds of Formula I can also be independently or in combination used as an antiviral, anticancer or an antitumor agent. These compounds can also be independently or in combination used with other antiviral, anticancer or an antitumor agents.
By xe2x80x9cantisensexe2x80x9d it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004).
By xe2x80x9c2-5A antisense chimeraxe2x80x9d it is meant, an antisense oligonucleotide containing a 5xe2x80x2 phosphorylated 2xe2x80x2-5xe2x80x2-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
By xe2x80x9ctriplex forming oligonucleotides (TFO)xe2x80x9d it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504).
By xe2x80x9cenzymatic nucleic acidxe2x80x9d it is meant a nucleic acid molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage and/or ligation of other nucleic acid molecules, cleavage of peptide and amide bonds, and trans-splicing. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. AU of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving activity to the molecule. The sites in such nucleic acid that can be modified with nucleotides described herein are known in the art, or standard methods can be used to determine useful such sites in other such nucleic acids. See, e.g., Usman, supra.
By xe2x80x9cenzymatic portionxe2x80x9d or xe2x80x9ccatalytic domainxe2x80x9d is meant that portion/region of the ribozyme essential for cleavage of a nucleic acid substrate (for example see FIG. 7).
By xe2x80x9csubstrate binding armxe2x80x9d or xe2x80x9csubstrate binding domainxe2x80x9d is meant that portion/region of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in FIGS. 1 and 3. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target together through complementary base-pairing interactions. The ribozyme of the invention may have binding arms that are contiguous or non-contiguous and may be varying lengths. The length of the binding arm(s) are preferably greater than or equal to four nucleotides; specifically 12-100 nucleotides; more specifically 14-24 nucleotides long. If a ribozyme with two binding arms are chosen, then the length of the binding arms are symmetrical (ie., each of the binding arms is of the same length; e.g., six and six nucleotides or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides or three and six nucleotides long).
In a preferred embodiment, a polynucleotide of the invention would bear one or more 2xe2x80x2-hydroxylamino functionalities attached directly to the monomeric unit or through the use of an appropriate spacer. Since oligonucleotides have neither aldehyde nor hydroxylamino groups, the formation of an oxime would occur selectively using an oligo as a polymeric template. This approach would facilitate the attachment of practically any molecule of interest (peptides, polyamines, coenzymes, oligosaccharides, lipids, etc.) directly to the oligonucleotide using either aldehyde or carboxylic function in the molecule of interest. 
Advantages of Oxime Bond Formation:
The oximation reaction proceeds in water
Quantitative yields
Hydrolytic stability in a wide pH range (5-8)
The amphoteric nature of oximes allows them to act either as weak acids or weak bases.
Oximes exhibit a great tendency to complex with metal ions
In yet another preferred embodiment, the aminooxy xe2x80x9ctetherxe2x80x9d in oligonucleotides, such as a ribozyme, is reacted with different compounds bearing carboxylic groups (e.g. aminoacids, peptides, xe2x80x9ccapxe2x80x9d structures, etc.) resulting in the formation of oxyamides as shown below. 
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings will first briefly be described.